1. Volume 69, Issue 2, Pages 257-265 (January 2006)
Butyrate modulates TGF-β1 generation and function: Potential renal benefit for Acacia(sen) SUPERGUM™ (gum arabic)? 
N. Matsumoto, S. Riley, D. Fraser, S. Al-Assaf, E. Ishimura, T. Wolever, G.O. Phillips, A.O. Phillips 
Kidney International 
Volume 69, Issue 2, Pages (January 2006)
DOI: /sj.ki
Copyright © 2006 International Society of Nephrology Terms and Conditions

2. Figure 1
Dietary supplementation with SUPERGUM™ increases serum butyrate. At the end of a 10-week study period in which normal healthy volunteers were fed 25 g/day of SUPERGUM™, serum samples were collected and (a) butyrate or (b) propionate were quantified as detailed in Materials and Methods. Data represent median and inter-quartile range; for butyrate n=7 and for propionate n=10.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2006 International Society of Nephrology Terms and Conditions

3. Figure 2
Butyrate suppresses basalTGF-β1 production by tubular epithelial cells. Increasing doses of butyrate were added to confluent monolayers of serum-deprived HK2 cells under serum-free conditions. Cell culture supernatant samples were collected after (a) 24, (b) 48, and (c) 72 h and TGF-β1 was quantified by ELISA. Data represent mean±s.d.; n=6. *P<0.05 compared to serum-free medium in the absence of butyrate.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2006 International Society of Nephrology Terms and Conditions

4. Figure 3
Propionate has no effect on basal TGF-β1production by tubular epithelial cells. Increasing doses of propionate were added to confluent monolayers of serum-deprived HK2 cells under serum-free conditions. Cell culture supernatant samples were collected after (a) 24, (b) 48, and (c) 72 h and TGF-β1 was quantified by ELISA. Data represent mean±s.d.; n=6. P>0.05 for all samples compared to serum-free medium in the absence of propionate.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2006 International Society of Nephrology Terms and Conditions

5. Figure 4
Butyrate-mediated suppression of TGF-β1 is associated with suppression of ERK activation. Increasing doses of either (a) the MEK inhibitor PD98059 or (b) p38 MAP kinase inhibitor SB were added to confluent monolayers of HK-2 cells under serum-free conditions for 24 h. Subsequently, cell culture supernatant samples were collected for analysis of TGF-β1 concentration by ELISA. Data represent mean±s.d.; n=3. *P<0.05 compared to serum-free medium alone in the absence of inhibitor. In parallel experiments, the effect of butyrate on ERK activation was examined by immunoblot analysis using antibodies to either dually phosphorylated active form of MAPK (p44/ERK1 and p42/ERK2) or total MAPK. (c) To determine the dose-dependent effect of SCFA, butyrate or propionate at the concentrations shown was added to confluent HK2 cell monolayers in the absence of serum for 30 min. (d) Time-dependent effects of butyrate were examined by addition of 1 mM butyrate to confluent monolayers of HK2 cells for up to 30 min. Cell lysis and immunoblot analysis were carried out as detailed in Materials and Methods.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2006 International Society of Nephrology Terms and Conditions

6. Figure 5
Butyrate suppresses 25 mM D-glucose-stimulated TGF-β1 synthesis. (a) Confluent monolayers of HK2 cells were stimulated with 25 mM D-glucose either alone or in the presence of butyrate (▪) or propionate () at the concentrations indicated for a total of 7 days under serum-free conditions. In control experiments, serum-free medium containing 5 mM D-glucose () was added to the cells. TGF-β1 concentration in the cell culture supernatant was examined by ELISA. (b) Data represent mean±s.d.; n=6, *P<0.05 compared to 25 mM D-glucose alone. In parallel experiments, de novo TGF-β1 protein synthesis was examined by stimulation of cells with 25 mM D-glucose either alone or in the presence of butyrate or priopionate in tissue culture medium to which 40 μCi of 3H-radiolabelled amino-acid mixture was added. Following 7 days of stimulation, TGF-β1 in the cell culture supernatant was immunoprecipitated and visualized by autoradiography.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2006 International Society of Nephrology Terms and Conditions

7. Figure 6
Butyrate-mediated suppression of 25 mM D-glucose-stimulated TGF-β1 is associated with suppression of ERK activations. (a) Increasing doses of either the MEK inhibitor PD98059 or (b) p38 MAP kinase inhibitor SB were added to confluent monolayers of HK-2 cells stimulated with 25 mM D-glucose under serum-free conditions for 24 h. Subsequently, cell culture supernatant samples were collected for analysis of TGF-β1 concentration by ELISA. Data represent mean±s.d.; n=3. *P<0.05 compared to serum-free medium alone in the absence of inhibitor. In parallel experiments, the effect of butyrate on 25 mM D-glucose-stimulated ERK activation was examined by immunoblot analysis using antibodies to either dually phosphorylated active form of MAPK (p44/ERK1 and p42/ERK2) or total MAPK. (c) To demonstrate activation of ERK, HK2 cells were stimulated with 25 mM D-glucose under serum-free conditions for up to 180 min. At the time points indicated, cells were lysed and ERK activation was assessed. (d) To determine the effect of butyrate, HK-2 cells were stimulated with 25 mM D-glucose either alone or in the presence of 1 mM butyrate for 15 min before cell lysis and immunoblot analysis.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2006 International Society of Nephrology Terms and Conditions

8. Figure 7
Antagonism of TGF-β1-mediated increase of SMAD-dependent signalling. HK-2 cells were transfected with a SMAD-responsive promoter (SBE)4-Lux, before stimulation with TGF-β1 (1 ng/ml) either alone or in the presence of either (a) butyrate or (b) propionate. All stimuli were applied for 6 h, subsequently luciferase content was quantified as described in Materials and Methods and the results were normalized for transfection efficiency (using β-galactosidase) expressed as the fold increase above the non-stimulated control. Data represent mean±s.d.; n=6.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2006 International Society of Nephrology Terms and Conditions

9. Figure 8
Suppression of Smad phosphorylation and distribution of TGF-β receptors. (a) Confluent monolayers of HK2 cells were stimulated with TGF-β1 (1 ng/ml) either alone or in the presence of increasing doses of either butyrate or propionate as indicated. Subsequently, 6 h after stimulation, cells were lysed and Smad phosphorylation was assessed by immuoblot analysis using an antibody. (b) TGF-β receptor distribution in lipid raft and non-raft fractions was analysed in HK-2 cells following sucrose gradient subcellular fractionation to separate lipid rafts from other cellular components. Confluent monolayers were stimulated with TGF-β1 (1 ng/ml) either alone or in combination with 1 mM butyrate for 24 h before cell lysis and fractionation as described in Materials and Methods. In control experiments, cells were exposed to serum-free medium alone. Membrane fractions were subsequently analysed by TGF-β RI immunoblot analysis. (c) Following scanning densitometry of autoradiographs, the distribution of TGF-β receptor into the raft (fractions 3–6) and non-raft (fractions 7–12) was quantified and the data from three separate experiments were expressed graphically and expressed as the percentage of the receptor in the raft-and non-raft-associated membrane fractions.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2006 International Society of Nephrology Terms and Conditions